The role of pioneer neurons in the development of mouse visual cortex and corpus callosum

The primordial plexiform neuropil is very critical to neocortical development. The pioneer neurons, mainly Cajal-Retzius cells in the marginal zone, and subplate neurons in the subplate, differentiate from the primordial plexiform neuropil. In this study, the development of corpus callosum, visual cortex, and subcortical pathways has been observed in C57BL/6 mice with various methods, such as DiI labeling in vitro and in vivo, DiI and DiA in vitro double labeling, immunocytochemistry, and in vivo BrdU and Fast Blue labeling. As early as E14, the primordial plexiform neuropil can be found in the telencephalic wall, and it contains many pioneer neurons. On E15 the primordial plexiform neuropil differentiates into the marginal zone and the subplate. Cajal-Retzius cells exist in the marginal zone, and subplate neurons are in the subplate. Either Cajal-Retzius cells or subplate neurons have long projections toward the ganglionic eminence, suggesting that they migrate tangentially from the ganglionic eminence. Cajal-Retzius cells are involved in radial migration, and subplate neurons participate to guide pathfinding of subcortical pathways. This study reveals how the pioneer neurons, through radial and tangential migration, play an important role in neocortical formation and in the pathfinding of the corpus callosum and subcortical pathways. Furthermore, DiI labeling in vivo has demonstrated the presence of pioneer neurons all along the corpus callosum pathway, especially in the midline. This suggests that pioneer neurons may also play a role in guiding the pathfinding of the corpus callosum. Accepted: 31 July 2001     

Preparation and characterization of porous beta-tricalcium phosphate/collagen composites with an integrated structure

Porous beta-tricalcium phosphate (TCP)/collagen composites with different beta-TCP/collagen weight ratio were prepared. The influences of the preparation conditions on the microstructure of porous composite and the joint status of beta-TCP particles with collagen fibrils were characterized by X-ray diffractometer, scanning electron microscopy and transmission electron microscopy. The results showed: (1) an acid treatment could effectively disassemble collagen fibrils; (2) in the resulting porous composites, beta-TCP particles homogenously existed on the skeleton of the collagen fibril network and bonded tightly to both the fibrils and themselves. The tight bonding formation could be due to the reaction between Ca ions in the particles and carboxyl groups in collagen polypeptide chains and due to the reprecipitation of partially dissolved beta-TCP during synthesis. The tight bonding between beta-TCP particles and collagen fibrils in the composites demonstrated an integrated structure, which was reproducible when beta-TCP/collagen ratio ranged from 2 to 4. Such integrated structure would make significant contributions in reliably tailoring properties of the porous composites by varying beta-TCP content. In addition, the porous composites had large porosity (approximately 95%) and appropriate pore size (approximately 100 microm), showed no negative impact in cytotoxicity assay and complete bone tissue regeneration after 12 weeks in animal test.     

生后人肺神经内分泌细胞的形态学观察—免疫组织化学及电镜研究

用银染色法、免疫组织化学及透射电镜技术，对３６例人肺的神经内分泌细胞进行了形态学和免疫组织化学观察。提示ＮＥ细胞发生了废用性退化，其原因可能与出生前后人肺功能改变有关。     

Anticocaine catalytic antibodies

Cocaine mediates its reinforcing and toxic actions through a "loss of function" effect at multiple receptors. The difficulties inherent in blocking a pleiotropic blocker pose a great obstacle for the classical receptor-antagonist approach and have contributed to the failure (to date) to devise specific treatments for cocaine overdose and addiction. As an alternative, we have embarked on an investigation of catalytic antibodies, a programmable class of artificial enzyme, as "peripheral blockers" -- agents designed to bind and degrade cocaine in the circulation before it partitions into the central nervous system to exert reinforcing or toxic effects. We synthesized transition-state analogs of cocaine's hydrolysis at its benzoyl ester, immunized mice, prepared hybridomas and developed the first anticocaine catalytic antibodies with the capacity to degrade cocaine to nonreinforcing, nontoxic products. We subsequently identified several families of anticocaine catalytic antibodies and found that the most potent antibody possessed sufficient activity to block cocaine-induced reinforcement, organ dysfunction and sudden death in rodent models of addiction, toxicity and overdose, respectively. With the potential to promote cessation of use, prolong abstinence and provide a treatment for acute overdose, the artificial enzyme approach comprehensively responds to the problem of cocaine.     

IL-1β诱导体外培养的大鼠中脑黑质神经元c-jun表达

应用细胞原位杂交技术，观察经重组小鼠白细胞介素-19（IL-1β）处理后的体外培养的新生1d大鼠中脑黑质神经元c－jun基因的表达．结果显示，培养的黑质细胞多为酪氨酸羟化酶阳性神经元，IL-1β可诱导体外培养的黑质神经元c－junmRNA表达，高水平的表达出现在IL-1β处理后2～4h。说明IL－1β有兴奋黑质神经元的作用，并提示黑质神经元上可能存在IL－1β受体．     

人胚胎肺内神经内分泌细胞电镜观察

用透射电镜对不同胎龄的人胚胎肺内神经内分泌细胞进行了发生和超微结构观察。肺内支气管上皮内未分化细胞，第八周开始向神经内分泌细胞转化，提示神经内分泌细胞对早期胚胎肺的发生、发育有特殊意义。神经内分泌细胞内致密核心小泡（ＤＣＶ）及其它与内分泌活动有关的各种细胞器发达。能见到Ｐ０细胞、Ｐ１细胞、Ｐ２细胞及Ｐ３细胞等四种类型的神经内分泌细胞。Ｐ０细胞的发生及分化程度表明它可能是一种前细胞，它可以进一步转化     

Recombinant single-chain variable fragment antibodies directed against Clostridium difficile toxin B produced by use of an optimized phage display system

Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C. difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10(-8) M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.     

Age-dependent DNA methylation changes in the ITGAL (CD11a) promoter

DNA methylation patterns change with age in a complex fashion, typically with an overall decrease in genomic deoxymethylcytosine (d(m)C) content, but with local increases in some promoters that contain GC-rich sequences known as CpG islands. While the consequences of age-dependent CpG island methylation have recently been studied in organs such as the colon, less is known about the functional significance of the progressive hypomethylation of promoters lacking CpG islands, and the significance of age-dependent changes in T cell DNA methylation is completely unexplored. We asked if age-dependent DNA hypomethylation might contribute to overexpression of the T cell ITGAL gene, which encodes CD11a, a subunit of LFA-1. CD11a mRNA increased with age as well as with experimentally induced DNA hypomethylation. This increase correlated with hypomethylation of sequences flanking the ITGAL promoter in vitro and in aging. 'Patch' methylation of the region suppressed promoter function. DNA methyltransferases 1 and 3a also decreased with aging. These results indicate that hypomethylation of regions flanking the ITGAL promoter may increase CD11a expression, and suggest that age-dependent hypomethylation of promoters lacking CpG islands, perhaps due to decreased DNA methyltransferase expression, may be one mechanism contributing to increased T cell gene expression with aging.